全 文 ：Journal of Systematics and Evolution 46 (1): 23–31 (2008) doi: 10.3724/SP.J.1002.2008.07059
(formerly Acta Phytotaxonomica Sinica) http://www.plantsystematics.com
Significance of RT-PCR expression patterns of CYC-like genes in
Oreocharis benthamii (Gesneriaceae)
1,2Zhi-Yan DU 1Yin-Zheng WANG *
1(State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China)
2(Graduate University of Chinese Academy of Sciences, Beijing 100049, China)
Abstract We isolated and characterized two CYC-like genes from Oreocharis benthamii Clark (Gesneriaceae) in
order to investigate their expression patterns. Phylogenetic analysis demonstrates that the two CYC-like genes,
ObCYC1 and ObCYC2, belong to GCYC1 and GCYC2 clades in Gesneriaceae, respectively. Gene-specific
RT-PCR results show that they have different spatio-temporal expression patterns. ObCYC1 is mainly expressed in
the adaxial region of developing flowers as CYC is in Antirrhinum majus. The expression pattern of ObCYC1
demonstrates its basic function of controlling the adaxial floral organ development that is similar to that of CYC in
Antirrhinum, which is in correlation with their conserved function domains, i.e. TCP and R domains. However,
ObCYC1 is likely to repress the growth of both the adaxial petals and adaxial staminode. In addition, the weak-
ened expression of ObCYC1 near flowering is correlated with the flower morphology of Oreocharis which is
characteristic of weak zygomorphy. ObCYC2 exhibits a remarkable differentiation in protein sequence of TCP and
R domain in comparison with those of ObCYC1 and CYC. Correlatively, ObCYC2 is inactive in floral tissue. This
unique feature of ObCYC2 needs to be investigated by further comparative study both in expression and function
in order to understand its basic function and evolutionary pathway in Gesneriaceae and related families.
Key words CYCLOIDEA, expression pattern, floral symmetry, Gesneriaceae, Oreocharis benthamii.
Flower symmetry is an important character in
angiosperm evolution and pollination. Genetic con-
trols of flower symmetry have been extensively
investigated in Antirrhinum majus L. (Carpenter &
Coen, 1990; Luo et al., 1996, 1999; Almeida et al.,
1997). Antirrhinum flowers are strongly zygomorphic
in the wild type while lose or reduce floral dorsoven-
tral asymmetry in mutants. CYCLOIDEA (CYC) and
DICHOTOMA (DICH) have been known to be essen-
tial for the development of zygomorphic flowers
(Carpenter & Coen, 1990; Luo et al., 1996, 1999).
Both of these genes are required for fully bilateral
symmetry development but the role of CYC is more
important. They are different in the timing and space
of expression with partial redundancy in function (Luo
et al., 1996, 1999). CYC and DICH have similar
amino acid sequences and belong to the TCP gene
family, a large gene family named for TEOSINTE
BRANCHED1 (TB1) in Zea mays L., CYCLOIDEA
(CYC) in A. majus and the proliferating cell factor
(PCF) DNA-binding proteins in Oryza sativa L.
(Doebley et al., 1995; Luo et al., 1996; Kosugi &
Ohashi, 1997; Cubas et al., 1999a).
Evidence from Antirrhinum demonstrates that
CYC is the key developmental gene in controlling
floral dorsoventral asymmetry and one of the major
driving forces of morphological evolution (Carroll,
2000). Besides Antirrhinum, homologues of CYC have
been isolated from some groups in Lamiales sensu
lato to reveal the relationship between the molecular
evolution of these genes and the morphological shift-
ing of floral symmetry. These research works show
that duplication and functional divergence of
CYC-like genes may have contributed to the evolution
of floral symmetry within Lamiales s.l. (Cubas et al.,
1999b; Hileman & Baum, 2003; Hileman et al., 2003;
Reeves & Olmstead, 2003). Phylogenetic analyses
indicate that Lamiales s.l. is a monophyletic group
believed to be ancestrally zygomorphic (Coen &
Nugent, 1994; Endress, 1998; Ree & Donoghue,
1999), and there is only a single ancestral CYC-like
gene at the base of Lamiales s.l. (Citerne et al., 2000;
Cubas, 2004).
As one of the most basal families in Lamiales s.l.,
Gesneriaceae is an ideal candidate for studying the
floral symmetry shifting because of its high proportion
of genera with actinomorphic flowers (Endress,
1998; Cubas, 2004). Some studies show that bilateral
symmetry is the ancestral form in Gesneriaceae and
radially symmetric flowers have been derived several
times from different clades with bilaterally symmetric
flowers (Smith, 1996, 2000; Smith et al., 1997; Cronk
& Möller, 1997; Möller et al., 1999). Even though

———————————
Received: 17 April 2007 Accepted: 12 December 2007
* Author for correspondence. E-mail: .
Journal of Systematics and Evolution Vol. 46 No. 1 2008 24
some CYC-like genes (GCYC) have been isolated
from both subfamilies Gesnerioideae and Cyrtan-
droideae in Gesneriaceae, the sequences used for
phylogenetic analyses are only 60%–70% of open
reading frame (ORF) (Möller et al., 1999; Wang et al.,
2004; Smith et al., 2004). Moreover, there is still no
data in expression and functional analysis of CYC-like
genes reported to date in Gesneriaceae. Research in
gene expression not only provides us with the exact
location of gene products, but also enables us to
understand their functional transition and develop-
mental homology among related genes or plant
groups, because expression differentiation is an
important content of gene evolution (Kellogg, 2004).
Therefore, we select Oreocharis benthamii, a repre-
sentative with weak zygomorphy predominant in
Gesneriaceae, to perform molecular and expression
studies of its CYC-like genes. In addition, since zy-
gomorphy is the ancestral form in Gesneriaceae, the
expression of CYC-like genes in Oreocharis may
represent a basic expression pattern and indicate a
basic function of such genes in the establishment of
floral symmetry in Gesneriaceae, which would shed
light on the study of the evolutionary developmental
pathway of CYC-like genes in Lamiales s.l.
1 Material and methods
1.1 Plant material
Leaves and flower buds of Oreocharis benthamii
C. B. Clarke, used in this study, were collected from
Dinghu Mountain, Zhaoqing City, Guangdong Prov-
ince, P. R. China.
1.2 Isolation and characterization of CYC-like
genes in O. benthamii
Genomic DNA was prepared from dried leaves
following a modified CTAB procedure (Doyle &
Doyle, 1987). Approximately 70% of uninterrupted
ORF of CYC-like genes was amplified using these
primers: forward GCYCFS (5′-ATGCTAGGTTTC-
GACAAGCC-3′) and reverse GCYCR (5′-ATGAA-
TTTGTGCTGATCCAAAATG-3′) (Möller et al.,
1999). Then we designed a new forward primer F1
(5′-ATGTTTGGAAAGAGCCCATAC-3′) in the
initiative position of the gene ORF according to the
sequences of CYC in A. majus L. and Linaria vulgaris
L. to amplify the upstream region of CYC-like ORF.
Total RNA was extracted from flower buds of O.
benthamii with the Plant RNA Purification Reagent
(Invitrogen, Carlsbad, CA, USA), and then mRNA
was isolated from total RNA using Oligotex mRNA
Mini Kit (Qiagen, Valencia, CA, USA). cDNA was
reverse transcribed with RevertAidTMH Minus First
Strand cDNA Synthesis Kit (Fermentas, EU) for
reverse transcriptase-polymerase chain reaction (RT-
PCR) analysis. The PCR products were cloned into
the pGEM-Teasy vector (Promega, USA) and se-
quenced. These genes were independently cloned at
least twice. We used DAMBE software (Xia & Xie,
2001) to translate the cDNA sequences of CYC-like
genes in O. benthamii. The amino acid sequences
were further compared with related homologues using
DNAMAN 5.29 (Lynnon Biosoft Company, USA)
and BioEdit v7.0.4 (Hall, 1999).
1.3 Phylogenetic analysis of CYC-like genes in O.
benthamii
Besides CYC-like genes from O. benthamii, we
selected CYC-like genes from some related taxa in
Gesneriaceae (Table 1), using CYCLOIDEA from A.
majus and LCYC from Linaria vulgaris as outgroups
(Table 1), to conduct a phylogenetic analysis.
ClustalW was used for the multiple alignment of the
amino acid sequences (Thompson et al., 1994), and
the amino acid matrix was optimized manually. This
amino acid matrix was analysed by PAUP*4.0b10 to
construct maximum parsimony (MP) trees under
default settings (Swofford, 2000). The bootstrap
values were calculated using 1000 replicates.
1.4 RT-PCR of CYC-like transcripts in O. ben-
thamii
We used two stages of O. benthamii flowers for
RT-PCR. One stage was middle stage (after petal
edges had been defined but before bud opening). The
other stage was late stage (petals were already open).
Flowers of these two stages were dissected into four
regions: sepals (removed from the outer whorl),
abaxial petal with corresponding corolla tube and
attached abaxial stamens, lateral petals with corre-
sponding corolla tube and attached lateral stamens,
and adaxial petals with corresponding corolla tube and
the attached staminode.
Total RNA was extracted from the tissue of the
four floral regions at the two stages of developing
flowers, respectively, for RT-PCR experiments.
RT-PCR was performed using gene-specific primers:
ObCYC1 (forward 5′-GTACTGCATCAACTCTAGC-
3′, reverse 5′-CGACACCTATTGATGAACTTG-3′),
ObCYC2 (forward 5′-GAGGACACTTTGTAAGCA-
3′, reverse 5′-GGTACTTCGCAAACTTCTG-3′).
The specificity of these primers was tested by
amplification of vectors with/without the special
segment insertion. Actin was used as a positive con-
trol (Prasad et al., 2001). The following thermocycling
conditions were employed: initial denaturation at 96
DU & WANG: Expression patterns of CYC-like genes in Oreocharis benthamii

25
℃ for 3 min; 26–30 cycles of 96 ℃ for 30 s, 55–60
℃ for 30 s, and 72 ℃ for 1 min; final extension at
72 ℃ for 10 min. The amplified products were
separated on a 1.5% agarose gel and the density of
ethidium bromide-stained bands was determined using
a Bioimaging System (Gene Tools Program; Syngene,
UK).


Table 1 Accession numbers for the CYC-like genes from Veronicaceae and Gesneriaceae examined in this study


2 Results
2.1 Sequence analysis
We isolated two CYC-like genes from O. ben-
thamii genomic DNA, and designated them as Ob-
CYC1 and ObCYC2 according to the result of phy-
logenetic analysis (Fig. 2). The ORF of ObCYC1 is
1004 bp long. Since we failed to clone ObCYC2 from
cDNA pool, the ORF of ObCYC2 is not complete,
with a length of 995 bp. Furthermore, we used BioEdit
v7.0.4 (Hall, 1999) to analyze protein sequences of
ObCYC1, ObCYC2, BlCYC1, BlCYC2 and CYC
(AmCYC) (Table 2; Fig. 1). In the three conserved
domains, i.e. TCP domain, R domain and ECE motif,
there are several amino acid substitutions of ObCYC
relative to CYC. Two substitutions are located in basic
region (S-C, Y-V) and one is in helix II region (D-E)
of TCP domain (Fig. 1). Beyond B-H-L-H region,
there are also four amino acid substitutions in TCP
domain among ObCYC, BlCYC and CYC. One is
between basic and helix I (I-M) region, another one is
between helix I and loop (G-A) regions, and two are
after helix II (T-V/A, V-I) region. The protein se-
quences of the R domain are conserved among Ob-
CYC1, BlCYC1 and CYC. ECE domain among Ob-
CYC, BlCYC and CYC is obviously divergent in amino
acid sequences (Fig. 1). The protein sequences in the
ORF are remarkably divergent in the non-conserved
regions between ObCYC and CYC and within ObCYC
(Fig. 1). In addition, there is a long insertion in Ob-
CYC after R domain relative to CYC (Fig. 1).
2.2 Phylogenetic analysis
Parsimony analysis of the amino acid matrix re-
sulted in a single most parsimonious tree (CI=0.74,
RI=0.79) (Fig. 2). Phylogenetic analysis (Fig. 2)
GenBank No. Taxon Gene name Reference
Y16313 Antirrhinum majus L. Cycloidea Luo et al., 1996
AF161252 Linaria vulgaris L. LCYC Cubas et al., 1999
AY423158 Didymocarpus citrina Ridl. GCYC1C Wang et al., 2004
AY423159 GCYC1D Wang et al., 2004
AY423161 Loxostigma sp. GCYC1C Wang et al., 2004
AY423162 GCYC1D Wang et al., 2004
AF208321 Conandron ramondioides Sieb. & Zucc. GCYC1 Citerne et al., 2000
AF208316 GCYC2 Citerne et al., 2000
AY423160 Cyrtandra apiculata C. B. Clarke GCYC1 Wang et al., 2004
AY423147 GCYC2 Wang et al., 2004
AF208322 Haberlea ferdinandi-coburgii Urum. GCYC1 Citerne et al., 2000
AF208317 GCYC2 Citerne et al., 2000
AF208331 Ramonda myconi Rchb. GCYC1 Möller et al., 1999
AF208318 GCYC2 Citerne et al., 2000
DQ064642 Saintpaulia ionantha B. L. Burtt. GCYC1A Wang et al., 2005
DQ064644 GCYC1B Wang et al., 2005
AF208328 Primulina tabacum Hance GCYC1 Möller et al., 1999
AF208340 Streptocarpus primulifolius Gand. GCYC1A Citerne et al., 2000
AF208336 GCYC1B Citerne et al., 2000
NA Bournea leiophylla W. T. Wang & K. Y. Pan GCYC1 Unpublished data
NA GCYC2 Unpublished data
NA Oreocharis benthamii C. B. Clarke ObCYC1 This study
NA ObCYC2 This study
Journal of Systematics and Evolution Vol. 46 No. 1 2008 26

Fig. 1. Alignment of the predicted amino acid sequences of ObCYC1, ObCYC2, BlCYC1, BlCYC2 and AmCYC. TCP, ECE and R domains are
lined out and the identical amino acids are in black boxes. TCP domain consists of basic, helix I, loop, and helix II motifs.


Table 2 The similarity values among CYC-like genes in Oreocharis benthamii, Bournea leiophylla and Antirrhinum majus (Data in the left column
show similarity on the nucleotide level and those in the right column show similarity on the amino acid level)
Gene ObCYC1 BlCYC1 ObCYC2 BlCYC2
BlCYC1 96.13% 92.54%
ObCYC2 68.79% 60.34% 68.11% 60.17%
BlCYC2 68.09% 59.49% 67.79% 59.32% 95.18% 90.94%
AmCYC 52.71% 50.29% 52.75% 50.72% 51.59% 47.06% 52.15% 46.92%


shows that each kind of GCYC is gathered together in
different clades, GCYC1 and GCYC2, which conforms
to previous phylogenetic trees (Möller et al. 1999;
Citerne et al., 2000; Wang et al., 2004). ObCYC1 and
ObCYC2, respectively, belong to GCYC1 and GCYC2
clades in Gesneriaceae. It should be stressed that both
ObCYC1 and ObCYC2 are more closely allied with
BlCYC1 and BlCYC2 from Bournea with nearly
actinomorphic flowers than any other GCYC in Ges-
neriaceae (Fig. 2).
DU & WANG: Expression patterns of CYC-like genes in Oreocharis benthamii

27


Fig. 2. Bootstrap 50% majority-rule consensus tree, showing the relationships among CYC-like genes of Oreocharis benthamii and the closely
related genera (CI = 0.74, RI = 0.79).


2.3 Locus-specific RT-PCR
The semiquantity gene-specific RT-PCR of O.
benthamii shows that the two copies of GCYC genes
in O. benthamii are different in expression. ObCYC1 is
mainly expressed in adaxial region of flowers while
ObCYC2 mRNA is not detectable in all floral tissues
(Fig. 3). Furthermore, the expression of ObCYC1 de-
clines near flowering relative to the mid-to-late stage.
Journal of Systematics and Evolution Vol. 46 No. 1 2008 28


Fig. 3. Gene-specific RT-PCR on RNA prepared from dissected Oreocharis benthamii flower buds. Dorsal, lateral and ventral corolla with
attached stamens dissected from middle stage and late stage buds. Sepal tissue is used as a negative control of ObCYC expression. Actin is used as
positive control. Gene specificity is confirmed by sequence analysis of RT-PCR products (MS, middle stage sepals; MD, middle stage floral tissue
from dorsal petals and staminode; ML, middle stage floral tissue from lateral petals and stamens; MV, middle stage floral tissue from ventral petals
and stamens; LS, late stage sepals; LD, late stage floral tissue from dorsal petals and staminodes; LL, late stage floral tissue from lateral petals and
stamens; LV, late stage floral tissue from ventral petals and stamens).


3 Discussion
3.1 Expression analysis of ObCYC1 and ObCYC2
Our RT-PCR results show that the two copies of
GCYC genes in O. benthamii are different in expres-
sion. ObCYC1 is mainly expressed in the adaxial
petals and staminode (Fig. 3), which is similar to that
of CYC in Antirrhinum. However, ObCYC1 is likely
to repress the growth of both the adaxial petals and
adaxial staminode, for the adaxial petals are smaller
than the two lateral and one abaxial petals in O.
benthamii. In addition, the expression of ObCYC1
declines obviously near flowering relative to the
mid-to-late stage, while CYC continues to be ex-
pressed at late stages in adaxial floral organs (Luo et
al., 1996). This difference indicates that the function
of ObCYC1 in controlling the adaxial part of flowers
might be weakened when flowers become mature as
suggested by its weakly zygomorphic corolla. In
addition, the adaxial petals of Antirrhinum are internal
asymmetry because of the expression of DICH re-
stricted to the most adaxial half of the adaxial petals,
which results in an internal asymmetry of the adaxial
petals (Luo et al., 1996, 1999). DICH-like genes have
been only found in Antirrhineae and probably spe-
cially contribute to the intrapetal differentiation of the
adaxial petals within Antirrhineae (Cubas, 2004).
Surprisingly, ObCYC2 mRNA is not detected in all
floral tissue (Fig. 3). Therefore, the internal symmetry
of adaxial petals in O. benthamii might be attributed
to symmetric expression of the single copy of ObCYC,
i.e., ObCYC1 in the adaxial petals unlike CYC sym-
metric versus DICH asymmetric expression in the
adaxial petals in Antirrhinum. In addition, one possi-
bility of ObCYC2 inactivity in the floral tissue is that
it may be a pseudogene, a common fate for duplicated
genes (Sankoff, 2001). Further work with in situ
hybridization and functional analysis is necessary with
respect to this point.
3.2 Protein sequence differentiation and func-
tional divergences of ObCYC
Changes of amino acid sequences usually lead to
the functional differentiation of duplicate genes, and
duplication of genes is a major mechanism for the
establishment of new gene functions and the genera-
tion of evolutionary novelties (Moore & Purugganan,
DU & WANG: Expression patterns of CYC-like genes in Oreocharis benthamii

29
2003). CYC-like genes usually have three conserved
domains possibly functioning in DNA binding and
protein-to-protein interactions, i.e., TCP domain, R
domain and ECE motif (Cubas et al., 1999a; Howarth
& Donoghue, 2006). Comparative analyses among
ObCYC, BlCYC and CYC show that in TCP domain
there exist three amino acid substitutions in ObCYC
and BlCYC relative to CYC (Fig. 1). Two substitutions
are located in basic region (S-C, Y-V) and one is in
helix II region (D-E) (Fig. 1). The basic region of TCP
domain is involved in the specificity of DNA binding
(Hurst, 1994; Littlewood & Evans, 1994; Kosugi &
Ohashi, 1997; Cubas et al., 1999a), while the role of
HLH region is not quite clear but one possibility is
that the helices mediate protein-protein interaction
through hydrophobic surfaces, in the same way as the
K domain of MADS box genes (Davies &
Schwarz-Sommer, 1994; Shore & Sharrocks, 1995).
The protein sequence differentiation in helix II of
ObCYC relative to CYC is located in a conserved
region, resembling the LXXLL motif, which is be-
lieved to mediate the interaction between TCP pro-
teins and liganded nuclear receptors (Heery et al.,
1997). Therefore, these three amino acid substitutions
in TCP domain may have contributed to the divergent
expression between ObCYC1 and CYC.
R domain, another conserved region, has consis-
tent protein sequence among ObCYC1, BlCYC1 and
CYC. The ECE motif is recently recognized between
TCP and R domain and it might be involved in stabi-
lizing the three-dimensional structure of proteins
(Howarth & Donoghue, 2006). ECE motif among
ObCYC, BlCYC and CYC is obviously divergent in
amino acid sequences (Fig. 1). However, it is highly
conserved between ObCYC1 and ObCYC2 as well as
between ObCYC and BlCYC in Gesneriaceae. Ob-
CYC2 has four additional amino acid substitutions in
TCP and R domains relative to GCYC1 and CYC. Two
substitutions take place within basic-helixI-loop
regions in TCP domain while another two in R do-
main. These substitutions merit further investigation
to test their effect on ObCYC2 function.
As outlined above, we conduct a comprehensive
comparative analyses between ObCYC and CYC,
which show that the protein sequence differentiations,
especially in the conserved domains, might be related
to the expression patterns of ObCYC, i.e., ObCYC1
and ObCYC2. The fact that ObCYC1 is mainly ex-
pressed in the adaxial petals and staminode as CYC in
Antirrhinum is probably due to their conserved TCP
and R function domains. In addition, the weakened
expression of ObCYC1 near flowering is correlative
with the flower morphology of Oreocharis which is
characteristic of weak zygomorphy. Since plants of
Gesneriaceae are characteristic of predominant weak
zygomorphy, the expression of ObCYC1 may repre-
sent the basic pattern of GCYC expression in control-
ling the adaxial parts of flowers in Gesneriaceae.
Furthermore, BlCYC2 from Bournea exhibits a similar
situation to ObCYC2 in protein sequence. Therefore,
GCYC2 clade needs further comparative researches
both in expression and function in order to understand
their basic function and evolutionary pathway in
Gesneriaceae and related families.
Acknowledgements This work was supported by
National Natural Science Foundation of China, Grant
nos. 30121003, 30570105.
References
Almeida J, Rocheta M, Galego L. 1997. Genetic control of
flower shape in Antirrhinum majus. Development 124:
1387–1392.
Carpenter R, Coen ES. 1990. Floral homeotic mutations
produced by transposon-mutagenesis in Antirrhinum
majus. Genes & Development 4: 1483–1493.
Carroll SB. 2000. Endless forms: The evolution of gene
regulation and morphological diversity. Cell 101:
577–580.
Citerne HL, Möller M, Cronk QCB. 2000. Diversity of
Cycloidea-like genes in Gesneriaceae in relation to floral
symmetry. Annals of Botany 86: 167–176.
Coen ES, Nugent J. 1994. Evolution of flowers and
inflorescences. Development (Suppl.): 107–116.
Cronk QCB, Möller M. 1997. Genetics of floral symmetry
revealed. Trends in Ecology & Evolution (Personal
edition) 12: 85–86.
Cubas P. 2004. Floral zygomorphy, the recurring evolution of a
successful trait. BioEssays 26: 1175–1184.
Cubas P, Lauter N, Doebley J, Coen E. 1999a. The TCP
domain: a motif found in proteins regulating plant growth
and development. The Plant Journal 18: 215–222.
Cubas P, Vincent C, Coen E. 1999b. An epigenetic mutation
responsible for natural variation in floral symmetry. Nature
401: 157–161.
Davies B, Schwarz-Sommer Z. 1994. Control of floral organ
identity by homeotic MADS-box transcription factors. In:
Nover L ed. Results and problems in cell differentiation.
Berlin: Springer-Verlag. 235–258.
Doebley J, Stec A, Gustus C. 1995. TEOSINTE BRANCHED 1
and the origin of maize: evidence for epistasis and the
evolution of dominance. Genetics 141: 333–346.
Doyle JJ, Doyle JL. 1987. A rapid DNA isolation procedure for
small quantities of fresh leaf tissue. Phytochemical
Bulletin 19: 11–15.
Endress PK. 1998. Antirrhinum and Asteridae-evolutionary
changes of floral symmetry. Symposia of the Society for
Experimental Biology 51: 133–140.
Hall TA. 1999. BioEdit: a user-friendly biological sequence
Journal of Systematics and Evolution Vol. 46 No. 1 2008 30
alignment editor and analysis program for Windows
95/98/NT. Nucleic Acids Symposium Series 41: 95–98.
Heery DM, Kalkhoven E, Hoare S, Parker MG. 1997. A
signature motif in transcriptional co-activators mediates
binding to nuclear receptors. Nature 387: 733–736.
Hileman LC, Baum DA. 2003. Why do paralogs persist?
Molecular evolution of CYCLOIDEA and related floral
symmetry genes in Antirrhineae (Veronicaceae).
Molecular Biology and Evolution 20: 591–600.
Hileman L, Kramer EM, Baum DA. 2003. Differential
regulation of symmetry genes and the evolution of floral
morphologies. Proceedings of the National Academy of
Sciences, USA 100: 12814–12819.
Howarth DJ, Donoghue MJ. 2006. Phylogenetic analysis of the
“ECE” (CYC/TB1) clade reveals duplications predating
the core eudicots. Proceedings of the National Academy of
Sciences, USA 103: 9101–9106.
Hurst HC. 1994. Transcription factors: 1 bZIP proteins. Protein
Profile 1: 123–168.
Kellogg EA. 2004. Evolution of developmental traits. Current
Opinion in Plant Biology 7: 92–98.
Kosugi S, Ohashi Y. 1997. PCF1 and PCF2 specially bind to
cis elements in the rice proliferating cell nuclear antigen
gene. Plant Cell 9: 1607–1619.
Littlewood J, Evans G. 1994. Transcription factors: 2 helix-
loop-helix. Protein Profile 1: 639–669.
Luo D, Carpenter R, Copsey L, Vincent C, Clark J, Coen E.
1999. Control of organ asymmetry in flowers of
Antirrhinum. Cell 99: 367–376.
Luo D, Carpenter R, Vincent C, Copsey L, Coen E. 1996.
Origin of floral asymmetry in Antirrhinum. Nature 383:
794–799.
Möller M, Clokie M, Cubas P, Cronk QCB. 1999. Integrating
molecular phylogenies and developmental genetics: a
Gesneriaceae case study. In: Hollingsworth PM, Bateman
RM, Gornall RJ eds. Molecular systematics and plant
evolution. London: Taylor and Francis. 375–402.
Moore RC, Purugganan MD. 2003. The early stages of
duplicate gene evolution. Proceedings of the National
Academy of Sciences, USA 100: 15682–15687.
Prasad K, Sriram P, Kumar CS, Kushalappa K, Vijayraghavan
U. 2001. Ectopic expression of rice OsMADS1 reveals a
role in specifying the lemma and palea, grass floral organs
analogous to sepals. Development Genes and Evolution
211: 281–290.
Ree RH, Donoghue MJ. 1999. Inferring rates of change in
flower symmetry in asterid angiosperms. Systematic
Biology 48: 633–641.
Reeves PA, Olmstead RG. 2003. Evolution of the TCP gene
family in Asteridae: cladistic and network approaches to
understanding regulatory gene family diversification and
its impact on morphological evolution. Molecular Biology
and Evolution 20: 1997–2009.
Sankoff D. 2001. Gene and genome duplication. Current
Opinions in Genetics and Development 11: 681–684.
Shore P, Sharrocks AD. 1995. The MADS-box family of
transcription factors. European Journal of Biochemistry/
FEBS 229: 1–13.
Smith JF. 1996. Tribal relationships within the Gesneriaceae: a
cladistic analysis of morphological data. Systematic
Botany 21: 497–514.
Smith JF. 2000. Phylogenetic signal common to three data sets:
combining data which initially appear heterogeneous.
Plant Systematics and Evolution 221: 179–198.
Smith JF, Hileman LC, Powell MP, Baum DA. 2004. Evolution
of GCYC, a Gesneriaceae homolog of CYCLOIDEA,
within Gesnerioideae (Gesneriaceae). Molecular
Phylogenetics and Evolution 31: 765–779.
Smith JF, Wolfram JC, Brown KD, Carroll CL, Denton DS.
1997. Tribal relationships in the Gesneriaceae: evidence
from DNA sequences of the chloroplast gene ndhF.
Annals of the Missouri Botanical Garden 84: 50–66.
Swofford DL. 2000. PAUP* Phylogenetic analysis using
parsimony (and other methods), version 4.0b10.
Sunderland, Massachusetts: Sinauer Associates.
Thompson JD, Higgins DG, Gibson TJ. 1994. CLUSTAL W:
improving the sensitivity of progressive multiple
alignment through sequence weighting, positions-specific
gap penalties and weight matrix choice. Nucleic Acid
Research 22: 4673–4680.
Wang CN, Möller M, Cronk QCB. 2004. Phylogenetic position
of Titanotrichum oldhamii (Gesneriaceae) inferred from
four different gene regions. Systematic Botany 29: 407–
418.
Wang L (王磊), Gao Q (高秋), Wang Y-Z (王印政), Lin Q-B
(林启冰). 2006. Isolation and sequence analysis of two
CYC-like genes, SiCYC1A and SiCYC1B, from
zygomorphic and actinomorphic cultivars of Saintpaulia
ionantha (Gesneriaceae). Acta Phytotaxonomica Sinica
(植物分类学报) 44: 353–361.
Xia X, Xie Z. 2001. DAMBE: Data analysis in molecular
biology and evolution. Journal of Heredity 92: 371–373.





DU & WANG: Expression patterns of CYC-like genes in Oreocharis benthamii

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苦苣苔科大叶石上莲 CYC类基因 RT-PCR表达模式研究
1, 2杜志岩 1王印政*
1(系统与进化植物学国家重点实验室, 中国科学院植物研究所 北京 100093)
2(中国科学院研究生院 北京 100049)

摘要 CYC 类基因的分子系统学研究已经在苦苣苔科Gesneriaceae中展开, 但是还缺乏对这些基因表达和功能的研究。因此,
我们选择苦苣苔科大叶石上莲Oreocharis benthamii作为实验材料, 分离出了CYC类基因的两个拷贝, 经过分子系统学分析这
两个基因分别属于苦苣苔科GCYC1和GCYC2两个分支, 故命名为ObCYC1和ObCYC2。分区的RT-PCR实验结果显示这两个基
因拥有不同的时间空间表达模式。ObCYC1与模式植物金鱼草Antirrhinum majus中的CYC基因类似, 集中在花冠背部区域表
达, 这与它们拥有保守的功能区TCP和R相一致。但是, ObCYC1与CYC表达模式仍有区别, 即, 和CYC相比ObCYC1在花冠背
部区域的表达提前减弱。这可能和大叶石上莲花冠微弱的两侧对称性相关。另外, 由于大叶石上莲的背部花瓣较两侧和腹部
花瓣小, 因此, 在功能上ObCYC1可能起抑制背部花瓣生长作用而CYC基因则促进背部花瓣生长。与ObCYC1不同, ObCYC2的
保守功能区有更多的氨基酸位点变化, 而且在RT-PCR实验中也没有检测到它的表达。因此, 需要开展更深入的实验研究分析
ObCYC2的基本功能, 这将有助于了解GCYC2类基因在苦苣苔科及其近缘科中的功能和进化途径。
关键词 CYCLOIDEA基因; 表达模式; 花对称性; 苦苣苔科; 大叶石上莲